This competing renewal U01 application proposes an investigation of the impact of rapidly evolving substance use patterns on HIV prevention and HIV-associated co-morbidities. We propose to undertake this work in Vancouver, Canada, where, as in many areas in the US, exponential growth in synthetic opioid use has become a health crisis. Accordingly, we seek to characterize emerging drug use patterns and the associated impacts on engagement in HIV prevention and medication assisted treatments for opioid use disorder (MAT). We also aim to assess willingness to use, uptake of, and adherence to new and emerging HIV-focused biomedical interventions (e.g., pre-exposure prophylaxis [PrEP], microbicides). In Vancouver, PrEP has recently been made available at no cost to those at high risk of HIV acquisition, including people who inject drugs (PWID). We further aim to examine key HIV-associated co-morbidities, in particular pain and neurocognitive impairment, among a combined sample of HIV-negative and HIV-positive PWID and the related impacts on substance use patterns, engagement in HIV prevention and MAT. This project involves the continuation of the Vancouver Drug Users Study (V-DUS; n = 1800), which is one of the longest-running prospective cohort studies of community-recruited people who use illicit drugs (PWUD) internationally. The V-DUS is comprised of two nested cohorts following HIV-negative street-involved drug-using youth aged 14-26 years and HIV-negative adult PWID since 1996. Our study setting is a key innovative feature of the proposed work. Vancouver is a port city that continues to experience high availability and prevalent use of a wide range of illicit drugs. It is home to one of the most dramatic outbreaks of HIV among PWID and is now an epicenter of the ongoing synthetic opioid epidemic. In response, a range of pioneering policies and programs specific to illicit drug use have been introduced, and several of these are now being scaled up and/or considered in the US. For example, a broad set of MAT are available for virtually free of charge through the universal healthcare plan and in a low threshold manner, which allows us to examine MAT-related outcomes without the confounding effects of insurance schemes and programmatic barriers associated with high-threshold provision of MAT. The V-DUS is also a member of the C3PNO consortium and thereby ideally positioned to advance team science approaches. V-DUS has been highly productive and is a globally unique, valuable resource for advancing HIV research and training future HIV researchers. During the past grant cycle, >130 of >160 peer-reviewed publications have been led by >70 trainees. The V-DUS is also harmonized with an ongoing NIDA-funded cohort of HIV-positive PWUD and therefore a key control/comparison in work focused on HIV/AIDS among PWUD is possible. Given our environment and track record to date, we are well positioned to generate important scientific evidence on HIV prevention and HIV- associated co-morbidities thereby contributing to high and medium research priorities identified by the NIH Office of AIDS Research and informing efforts to end the HIV/AIDS pandemic.